WO1987000201A1 - Cellules epitheliales exprimant un materiau genetique etranger - Google Patents

Cellules epitheliales exprimant un materiau genetique etranger Download PDF

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WO1987000201A1
WO1987000201A1 PCT/US1986/001378 US8601378W WO8700201A1 WO 1987000201 A1 WO1987000201 A1 WO 1987000201A1 US 8601378 W US8601378 W US 8601378W WO 8700201 A1 WO8700201 A1 WO 8700201A1
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keratinocytes
genetic material
cells
foreign genetic
selectable marker
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Jeffrey R. Morgan
Richard C. Mulligan
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Whitehead Institute for Biomedical Research
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Whitehead Institute for Biomedical Research
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Priority to EP86904590A priority patent/EP0228458B2/fr
Priority to AT86904590T priority patent/ATE68013T1/de
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/61—Growth hormone [GH], i.e. somatotropin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/60—Materials for use in artificial skin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/635—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
    • A61L2300/254—Enzymes, proenzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/258—Genetic materials, DNA, RNA, genes, vectors, e.g. plasmids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/43—Hormones, e.g. dexamethasone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
    • A61L2300/64—Animal cells
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/948—Microorganisms using viruses or cell lines

Definitions

  • the skin is the largest organ in the human body and consists of two components, the epidermis and the dermis.
  • the dermis is a relatively inert structure which consists of collagen and other matrix materials.
  • the epidermis lies above the dermis and is separated from it by a basement membrane.
  • the epidermis undergoes constant cell renewal and is regenerated approximately every 26 days.
  • the major cellular constituent of the epidermis is the keratinocyte, which provides an environment for nonkeratinocytes (e.g., elanocytes, Langerhans cells, Merkel cells and various immunological cells) which also occur in the epidermis.
  • Keratinocytes are cells which produce keratin, an insoluble fibrous protein, and are able to form a stratified squamous epithelia. Like other cells in the body, keratinocytes contain an entire complement of all genetic material.
  • keratinocytes Only a small percentage of the genes contained in keratinocytes are, however, expressed at levels which are biologically func ⁇ tional; that is, most of the genes in keratinocytes are not expressed at all or are expressed at such low levels that the poplypeptides they encode are produced in undetectable amounts or concentrations which are biologically functional or significant.
  • corneal epithelia and ⁇ on- junctival epithelia are stratified squamous epi ⁇ thelia and the predominant cell in each of these tissues is the keratinocyte. Keratin is to a large degree responsible for the mechanical protective function of the epidermis.
  • the epi ⁇ dermis acts as a barrier layer which prevents toxic substances and microorganisms from entering the skin and water and electrolytes from being lost.
  • the epidermis consists of two major layers. Outermost is the stratum corneum, which is a laminated layer of anucleate cornified cells. Next is a succession of viable inner cell layers, re ⁇ ferred to as the malpighian layers from which the cornified cells arise.
  • the malpighian layers are the basal cell layer, the stratum spinosum and the stratum granulosum.
  • the basal cell layer which lies adjacent to the basement membrane, is the germinative layer in which the majority of cell division occurs.
  • the stratum spinosum is a layer of flattened nucleated cells having characteristic keratohyaline granules.
  • the stratum granulosum lies between the stratum spinosum and the stratum corneum and is considered transitional between the nucleated cells of the former and the anucleate cells of the latter.
  • the cells divide in the basal layer, they move upward and progress to the other epidermal layers.
  • the keratinocytes undergo changes in shape and cytoplasmic structure. These changes result in the viable, metabolically active cells being transformed into the anucleate, corni ⁇ fied cells of the horny layer; these cells consist of keratin filaments surrounded by a cross linked protein envelope.
  • Epidermal cells are considered to occur in proliferative units or columns.
  • the base of each column is a group of basal cells, which are classi ⁇ fied as peripheral or central according to whether they lie beneath the periphery or the center of the column.
  • the central basal cell divides; some of the resulting daughters in turn divide and move to peripheral basal positions.
  • the peripheral basal cells then progress up through the successive epidermal layers. They are transformed into keratinized squamous cells, which ultimately flake off and are lost from the body.
  • the central basal cells are stem cells. Descendants of these stem cells will not die throughout the individual's lifetime. These basal cells are immortal and each time they divide, an immortal daughter cell results.
  • the other daughter cells become differentiating cells and are ultimately shed from the body.
  • the epidermis is one of only a few tissues in the body which undergo constant cell renewal; these include other epi ⁇ thelia, such as the lining of the small intestine, and bone marrow.
  • the invention described herein is based on the introduction into epithetial cells of foreign genetic material or genetic material not normally expressed in biologically significant concentrations such cells.
  • Epithelial cells of this invention have incor ⁇ porated in them foreign genetic material and express the incorporated foreign genetic material.
  • the foreign genetic material can be DNA or RNA which does not occur in epithelial cells; DNA or RNA which occurs in epithelial cells but is not expressed in them at levels which are biologically significant (i.e., levels sufficient to produce the normal physiological effects of the polypeptide it en- codes) ; DNA or RNA which occurs in epithelial cells and has been modified so that it is expressed in epithelial cells; and any DNA or RNA which can be modified to be expressed in epithelial cells, alone or in any combination thereof.
  • epi- thelial cells of the present invention can express genetic material encoding a selectable marker by which cells expressing the foreign genetic material can be identified.
  • retroviral vectors have been used to incorporate the foreign genetic material and the genetic material encoding the selectable marker into epithelial cells, particularly keratinocytes. It is also possible to introduce foreign genetic material into other epithelial cells, such as cells of the cornea, the conjunctiva, the lining of the gastrointestinal tract, the lining of the vagina, and the trachea and into bone marrow cells. Ex ⁇ pression of these genes by the keratinocytes into which they have been incorporated has also been demonstrated.
  • a method of using retroviral vectors which have recombinant genomes to introduce the two types of genetic material into epithelial cells is also a subject of the present invention.
  • epithelial cells of the present invention there are many advantages to epithelial cells of the present invention which make them very useful.
  • an epidermis having kera ⁇ tinocytes of the present invention would actually synthesize the polypeptide (e.g., a hormone, enzyme, drug) encoded by the genetic material incorporated into it according to the present invention.
  • the epidermis would thus serve as a continuous delivery system for that polypeptide.
  • the often-encountered problem of patient compliance with a prescribed regimen would be avoided because the hormone or other polypeptide would be constantly diffused into the bloodstream.
  • an isolated polypeptide such- as insulin
  • it Before an isolated polypeptide, such- as insulin, can be injected into the body, it must be extensively purified and characterized. Using epithelia having keratinocytes modified according to the present invention, how ⁇ ever, once the gene has been isolated, it can be introduced into the cells, which will produce the polypeptide hormone as it would normally be pro- prised. (In the case of insulin, for example, as it would normally be produced in the pancreas.)
  • a graft having keratinocytes of the present invention is that by controlling the size of the graft, the amount of the polypeptide delivered to the body can be controlled.
  • it is a skin graft, it can be excised if there is no longer a need for the polypeptide being produced. For example, if delivery of the polypeptide (hormone, enzyme, or drug) is necessary only for a specific period, the engineered graft can-be removed when treatment is no longer needed.
  • Another important advantage of the delivery system possible as a result of this invention is that because it is a continuous delivery system, the fact that polypeptide hormones have very short half lives is not a drawback.
  • the half life of HGH is about 19 minutes and in the case of native insulin (pure insulin) it is about 3-4 minutes.
  • genes can be introduced into keratino ⁇ cytes using a retroviral vector, they can be "on" (subject to) the retroviral vector control; in such a case, the gene of interest is transcribed from a retroviral promoter.
  • a promoter is a specific nucleotide sequence recognized by RNA polymerase molecules that start RNA synthesis. It is possible to make retroviral vectors having promoter elements (in addition to the promoter incorporated in the recombinant retrovirus) which are responsible for the transcription of the gene.
  • heat shock proteins are proteins encoded by genes in which the promoter is regulated by temperature.
  • the promoter of the gene which encodes the metal- containing protein metallothionine is responsive to Cd ions. Incorporation of this promoter or another promoter influenced by external cues also makes it possible to regulate the production of the polypeptide by the engineered keratinocytes.
  • Figure 1 is a schematic representation of a typical murine leukemia virus (retroviral) genome.
  • Figure 2 is a schematic representation of a recombinant retroviral genome into which the neo gene has been incorporated.
  • Figure 3 is a block diagram of one embodiment of the method used to introduce a dominant select ⁇ able marker (the neo gene) into keratinocytes.
  • Figure 4 is a schematic representation of a recombinant retroviral ' genome having the neo gene arid a gene encoding human growth hormone (HGH) .
  • Figure 5 is a pictorial representation of one embodiment of the procedure used to produce, detach and transplant an epithelial sheet in which the keratinocytes have foreign genetic material and genetic material encoding a dominant selectable marker.
  • Figure 6 is a graph prepared according to Example 4 representing the quantity of parathyroid hormone secreted in the designated time periods.
  • the foreign genetic material can be DNA or RNA which does not occur in epithelial cells; DNA or RNA which occurs in epi ⁇ thelial cells but is not expressed in them at levels which are biologically significant (levels suffi ⁇ cient to produce the- normal physiological effects of the polypeptide it encodes); DNA or RNA which occurs in epithelial cells and has been modified so that it is expressed in epithelial cells; and any DNA or RNA which can be modified to be expressed in epithelial cells, alone or in any combination thereof epi- thelial cells of the present invention express foreign genetic material.
  • epithelial cells of the present invention can express genetic material encoding a selectable marker by which cells expressing the foreign genetic material can be identified.
  • foreign genetic material encoding a hormone can be introduced into keratinocytes by cocultivation of the keratinocytes with a feeder layer producing infectious virus in which there is a recombinant genome having the foreign genetic material.
  • the recombinant genome can also have genetic material encoding a dominant selectable marker.
  • keratinocytes can be co- cultivated with Psi am cells, which produce infectious viruses in which there is a recombinant genome having genetic material encoding human growth hormone.
  • Keratinocytes expressing the neo gene and the foreign genetic material encoding HGH that is, as a result of this invention, it is possible to make keratinocytes expressing a dominant selectable marker and a polypeptide not normally expressed by such cells at biologically significant levels.
  • Keratinocytes expressing the two types of genetic material can be grown to confluence; removed as an epithelial sheet from the culture vessel in which they were grown; and applied to the body.
  • the epithelial sheet can provide a continuous supply of the hormone, enzyme or drug made by the keratinocytes.
  • Green and co-workers have developed techniques which make it possible to grow human epidermal cells or other keratinocytes in cultures with fibroblast cells which have been treated to make them unable to multiply.
  • the presence of fibroblast cell products (supplied from medium harvested from fibroblast cultures) was shown by Green et al. to be essential to support growth of keratinocytes. Fibroblast cell density is controlled in these cultures to allow epidermal cell colony formation and growth.
  • Green and co-workers it is possible to serially culture human epidermal cells and greatly increase the number present in the primary culture.
  • a specific procedure for the cultiva ⁇ tion of keratinocytes involves disaggregation of epidermis into keratinocytes by means of an enzyme
  • fibroblast cells are able to attach to the dish and provide factors ' necessary for ker tinocyte growth, but are not themselves able to replicate.
  • the cocultivation is carried out in Dulbecco's modified Eagle's Media containing 10% fetal calf serum, as well as adenine, cholera toxin, hydrocortisone, transferrin, insulin and epidermal growth factor.
  • each colony initiated by a single cell forms a stratified epithelium.
  • Cell division occurs in a layer of basal cells (those adjacent to the bottom of the dish) . These basal cells are responsible for all cell multiplication and cells that leave the basal layer become ter ⁇ minally differentiated.
  • basal cells are responsible for all cell multiplication and cells that leave the basal layer become ter ⁇ minally differentiated.
  • Epidermal cells cultured in this fashion have the main cytological features of keratinocytes and the cells grown in culture are a reasonable approximation of the epidermis.
  • Retroviruses are RNA viruses; that is, the viral genome is RNA.
  • This genomic RNA is, however, reverse transcribed into a DNA intermediate which is integrated very efficiently into the chromosomal DNA of infected cells.
  • This integrated DNA .intermediate is referred to as a provirus.
  • the retroviral genome and the proviral DNA have three genes: the gag, the pol and the env, which are flanked by two long terminal repeat (LTR) sequences.
  • the gag gene encodes the internal structural (nucleocapsid) proteins; the pol gene encodes the RNA-directed DNA polymerase (reverse transcriptase) ; and the env gene encodes viral envelope glycoproteins.
  • the 5' and 3' LTRs serve to promote transcription and polyadenylation of virion Rl.As.
  • Adjacent to the 5' LTR are sequences necessary for reverse transcription of the genome (the tRNA primer binding site) and for efficient encapsidation of viral RNA into particles (the , or Psi, site) .
  • Mulligan, R.C. Construction of Highly Transmissible Mammalian Cloning Vehicles Derived from Murine Retroviruses, In: Experimental Manipulation of Gene Expression, M. ⁇ nouye (ed) , 155-173 (1983) ; Mann, R. , Mulligan R.C. and Baltimore, D., Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell, 33:153-159 (1983) ; Williams, D.A. et al. , Introduction of new genetic material into pluripotent haematopoietic stem cells of the mouse, Nature , 310:476-480 (1984).
  • pMOV-Psi ⁇ which is an ecotropic Moloney murine leukemia virus (Mo-MuLV) clone.
  • pMOV-Psi expresses all the viral gene products but lacks a sequence (the or Psi sequence) necessary for encapsidation- of the viral genome.
  • pMOV-Psi " expresses an ecotropic viral envelope glycoprotein which recognizes a receptor present only on mouse (and closely related rodent) cells.
  • Another cell line is the NIH Psi am line, which are Psi-2-like packaging cell lines.
  • Psi-am cell lines contain a modified pMOV ⁇ psi-genome in which the ecotropic envelope glycoprotein has been replaced with envelope sequences derived from the amphotropic virus 4070A. As a result, they are useful for production of recombinant virus with amphotropic host range.
  • the retrovirus used to make the Psi am cell line has a very broad mammalian host range (an amphotropic host range) and can be used to infect human cells. As long as the recombinant genome has the Psi packaging sequence, the Psi-am cell line is capable of packaging recombinant retroviral genomes into infectious retroviral particles.
  • the retroviral genome has been modified by Cone and Mulligan for use as a vector capable of introducing new genes into cells. As shown in Figure 2, the gag, the pol and the env genes have all been removed and a DNA segment encoding the neo gene has been inserted in their place. The neo gene serves as a dominant selectable marker.
  • the retroviral sequence which remains part of the recombinant genome includes the LTRs, the tRNA binding site and the Psi packaging site.
  • Cone, R. and Mulligan, R. High-efficiency gene transfer into mammalian cells: Generation of helper-free recombinant retrovirus with broad mammalian host range, Proceedings of the National Academy of Sciences, U.S.A. , ⁇ :6349-6353 (1984).
  • a cell line producing recombinant amphotropic retrovirus having a recombinant genome is used in cocultivation with keratinocytes.
  • the recombinant genome is comprised of two LTRs and, in place of the gag, the pol and the env sequences, a neo gene.
  • the Psi am cell line originally derived from a 3T3 cell line which can be modified using standard techniques to include the recombinant retroviral genome, has been deposited with the American Type Culture Collection (Rock- ville, MD) under deposit number CRL8859.
  • the neo gene is a bacterial gene derived from the transposon Tn5, which encodes neomycin resis- tance in bacteria and resistance to the antibiotic G418 in mammalian cells.
  • This neo gene acts as a dominant selectable marker; its presence in a mammalian cell converts the cell into one which will grow in the presence of G418. (In its absence, the cell dies in the presence of G418.)
  • the presence of this gene in a mammalian cell can be determined by selection for its presence in cells grown in media which contains G418.
  • the recombinant retrovirus having this recombinant genome is re- ferred to as the neo virus. •
  • the fibroblast cell line used as the feeder layer for (in cocultivation with) keratinocytes is a Psi am line producing the neo virus at relatively high titers (e.g., between 10 4 and 105 neo units per mil) .
  • the neo gene is introduced into keratinocytes by means of this retroviral vector and its presence in the keratinocytes verified.
  • the neo gene was introduced into keratinocytes according to the procedure represented in Figure 3.
  • the procedure is described in detail in Example 1.
  • Psi am line producing the neo virus was treated with mitomycin C, which is an antibiotic which crosslinks DNA and thus renders the cells unable to divide. They are, however, still capable of producing infectious virus.
  • the suspen ⁇ sion of keratinocytes was plated onto a dish containing the treated Psi am cells, which served as a feeder layer producing infectious virus.
  • this feeder layer is resistant to G418 (because it has been engineered to contain the neo gene) . Unlike the Psi am line, it does not produce infectious virus. As a result, it can serve as a feeder layer during the selection process. This is referred to as the G418 feeder layer.
  • the disaggregated keratinocytes were plated on this G418 resistant feeder layer and G418 was added to the culture. After incubation, those cells which were growing up were resistant to G418; there were no colonies growing up from control cells (unin- fected cells) . The G418 resistant cells appeared to be viable normal keratinocyte colonies, indicating that the neo gene had been introduced into and expressed in these cells via the retroviral vector.
  • the keratinocytes containing the neo gene are of the normal wild type. This has been demonstrated in several ways. For example, total cell protein extracted from * infected and uninfected cells; was fractionated electrophoretically and visualized. The protein profile of the infected cells was very distinctive of keratinocytes. There are four major keratin proteins in keratinocytes (58, 56, 50 and 46 Kd in size) and these were at normal levels, both quali ⁇ tatively and quantitatively, in the uninfected and the infected keratinocytes. Analysis by Western blotting demonstrated the presence of involucrin.
  • Involucrin is the precursor protein of the cross- linked envelope which is distinctive of terminal differentiation in keratinocytes and is therefore a distinctive terminal differentiation marker of keratinocytes.
  • the G418 resistant keratinocytes were also shown by electron microscopy to have the appearance of normal keratinocytes: that is, G418 resistant keratinocytes colonies were five or six cell layers thick and contained keratin filaments, tonofilaments, and numerous desmosomes—all hall- marks of normal keratinocyte colonies.
  • G418-resistant keratinocytes have also been grown to confluence, segregated as an intact epi ⁇ thelium and transplanted onto athymic or nude mice. This approach demonstrates the ability of the epidermis to continue to differentiate, a process only partially evident in tissue culture. This is carried out in the following manner which is described in greater detail in Example 3. G418 resistant keratinocytes were grown using the G418 feeder layer; growth continued until adjoining colonies fused and became confluent. The sheet of epidermal cells was lifted off the dish, at which point, it contracted to about half the size of the culture vessel. The epithelium was transplanted to an appropriate recipient, which in this case was an athymic or nude mouse. Because the athymic or nude mouse lacks a thymus, it is incapable of rejecting transplanted tissue.
  • the recombinant retroviral vectors having the neo gene also have a cloning site. This makes it possible to introduce foreign genetic material into the vector and to have it expressed by keratinocytes cocultivated with the recombinant retrovirus. At the Bam Hi cloning site, it is possible to insert 'foreign genetic material.
  • the foreign genetic material can be DNA or RNA which does not occur in epithelial cells; DNA or RNA which occurs in epi- thelial cells but is not expressed by them at levels which are biologically effective (i.e., levels sufficient to produce the normal physiological effects of the polypeptide it encodes) ; DNA or RNA which occurs in epithelial cells and has been modified so that it is expressed by epithelial cells; and any DNA or RNA which can be modified to be -expressed by epithelial cells, alone or in any combination thereof.
  • HGH human growth hormone
  • HGH is a polypeptide of about 29,000 Daltons which is normally secreted only by the hypothalamus. Although the HGH gene is present in keratinoc tes, it is not expressed in those cells at biologically significant levels. Keratinocytes capable of making a polypeptide hormone such as HGH, or another substance not normally made by such cells at bio ⁇ logically significant levels, can be transplanted onto an individual and serve as a continuous supply system for the hormone or other substance.
  • a Psi am line which produce ' s a recombinant retrovirus having the gene of interest — here, the human growth hormone gene — can be constructed.
  • a modified DNA segment encod ⁇ ing a cDNA of human growth hormone is ligated into the Bam HI site of plasmid DNA having a recombinant retroviral genome; in one embodiment, this is the neo gene.
  • the plasmid is isolated and transfected onto Psi am cell.
  • Psi am cells producing the HGH-neo recombinant virus construct, which is represented in Figure 4 are isolated as G418 resistant colonies.
  • the Psi am cells producing the HGH-neo recombi ⁇ nant virus construct are used as the feeder layer in cocultivation with disaggregated keratinocytes. After cocultivation with the Psi am cells producing the HGH-neo recombinant virus construct, the keratinocytes are allowed to grow to confluence; are disaggregated into keratinocytes; and cocultivated with the G418 resistant cell line as previously des ⁇ cribed.
  • keratinocytes which have been cocultivated with the recombinant retrovirus having a recombinant genome containing foreign genetic material, to express the foreign genetic material can be assessed using several techniques. These techniques are described in greater detail in Example 3. As a result, it has been demonstrated that keratinocytes are able to secrete a polypeptide hormone which is normally not secreted at bio ⁇ logically significant levels by keratinocytes and, additionally, that they can secrete this hormone at close to physiological concentrations.
  • Transplanta ⁇ tion of the epithelium which is secreting human growth hormone can ' be used to demonstrate whether it can secrete suffi ⁇ cient quantities to produce a systemic5effeet.
  • the manner in which this transplantation of epidermis having keratinocytes in which new genetic material (e.g., the neo gene and a selected gene encoding a polypeptide of interest) can be done is outlined in Figure 5. It is possible to make an epithelium, in which the keratinocytes have new genetic material, suit ⁇ able for grafting onto a recipient.
  • the recipient could be the original source of the epithelial cells or could receive a graft from an appropriately matched donor.
  • a section of epidermis is disaggregated into individual keratino ⁇ cytes, which are cocultivated with a Psi am line producing both the neo gene and the gene of inter ⁇ est.
  • G418-resistant keratinocytes are selected, grown to confluence and detached as an epithelium. The detached epithelium is subsequently grafted onto the recipient.
  • Genes encoding polypeptides other than HGH can also be introduced into keratinocytes by means of the retroviral vector.
  • genes encoding parathyroid hormone (PTH) and insulin have been introduced into keratinocytes along with the neo gene.
  • Parathyroid hormone is a polypeptide involved in the regulation of calcium in the body.
  • Insulin is a polypeptide which regulates glucose levels in the bloodstream.
  • each of these poly ⁇ peptide hormones requires processing of the poly ⁇ peptide before it can be active.
  • parathyroid hormone has a presequence and a pro- sequence at the ami*.o terminus of the protein.
  • Insulin is a peptide made of a and b chains which are connected by disulfide bonds.
  • the precursor to insulin contains another peptide, the c peptide.
  • this c peptide must be cleaved otit in order to yield active insulin molecules. To date, this cleavage has been shown to be carried out in pancreatic cells and possibly by neuron cells.
  • the His D gene can be used for this purpose.
  • the His D gene is a bacterial gene from Salmonella and encodes histidinol dehydrogenase, a polypeptide which converts histidinol to histidine. Histidine is an essential amino acid; histidinol is an alcohol analogue of histidine and can be converted to histidine under the proper metabolic conditions. If cells are grown in media containing histidinol but lacking histidine, those cells having the His D gene can convert histidinol to histidine. Because histidine is essential to their function, those cells which have the His D gene (and thus can make histidine) will survive and those lacking the gene will not.
  • a retrovirus vector having the His D gene has been used to infect keratinocytes.
  • the keratino- cytes containing His D gene were selected by growing these cells in media lacking histidine but contain ⁇ ing histidinol.
  • keratinocytes having the His D gene formed colonies and grew to conflu ⁇ ence; those lacking the gene .did not. In fact, such cells occurred at a much higher frequency than those in which the neo gene was included.
  • independent dominant selectable markers e.g., the neo gene and the His D gene
  • polypeptides which have two different subunits for example, separate dominant selectable markers could be used to introduce the genetic information encoding the two subunits.
  • two or more dominant selectable markers could be used in the case of polypeptides which need to be specifically cleaved or processed in order to become active (e.g., insulin and parathyroid hormone).
  • a gene encoding the necessary processing enzyme could be introduced along with the gene encoding the polypeptide hormone requiring such processing. This would enable keratinocytes to process that polypep ⁇ tide hormone.
  • Keratinocytes Having Foreign Genetic Material It is also possible to use vehicles other than retroviruses to genetically engineer or modify keratinocytes and other epithelial cells. New genetic information could be introduced into kera ⁇ tinocytes by means of any virus which can express the new genetic material in such cells. For ex ⁇ ample, SV40 , Herpes virus, Adeno virus and human papilloma virus could be used for this purpose. Human papilloma virus, which causes warts, may be particularly useful for at least three reasons. First, this virus naturally infects keratinocytes.
  • the genome is circular and in transformed cells, it remains circular and replicates extra- chromosomally (e.g., its DNA does not integrate into genomic DNA of transformed cells, as retroviral DNA does) .
  • a large number of copies (e.g. , between 50 and 200) of the viral DNA is made per transformed cell. This is particularly useful because transformed cells are therefore able to produce large quantities of the polypeptide encoded by the new genetic material (e.g., hormone, enzyme, etc.) .
  • the present invention makes it possible to genetically engineer keratinocytes capable of forming epithelia which can secrete products (e.g., clotting factors, im unoregulatable factors, and polypeptide hormones) into the bloodstream.
  • the epithelia formed in this way can serve as a con ⁇ tinuous drug delivery system to replace present regimens, which require periodic administration (by ingestion, injection, etc.) of the needed substance.
  • it could be used to provide continuous delivery of insulin, which at the present time, must be isolated from the pancreas, extensively purified and then injected into the body by those whose insulin production or utilization is impaired. In this way, insulin could be introduced into the body via a continuous drug delivery system and there would be no need for daily injections of insulin.
  • Genetically engineered epidermis can also be used for the production of clotting factors. Hemophili ⁇ acs lack a protein called Factor VIII, which is involved in clotting. Factor VIII is now adminis ⁇ tered by injection; keratinocytes having genes encoding Factor VIII, can be used to make epithelia and producing Factor VIII; as a skin graft, the tissue would secrete the factor into the blood ⁇ stream.
  • LHRH lutenizing hormone releasing hormone
  • Interleukin 2 and Interleukin 3 which stimulate the immune system, are potentially valuable in the treatment of AIDS and could be delivered by a skin graft having keratinocytes genetically engineered to produce these two polypeptides (which are now administered by periodic injection) .
  • Another use of genetically engineered kera ⁇ tinocytes is to change hair growth patterns in an individual or to grow hair in culture for trans ⁇ plantation. Because hair formation is genetically controlled and hair is an epidermal appendage, keratinocytes have potential use in changing hair growth. .
  • Another application is to improve the general properties of the skin.
  • the skin has several important properties. It provides a barrier to the external surface, is a highly elastic structure, is resilient and has very excellent protective quali ⁇ ties.
  • the recombinant retroviral vector of this invention could be used to introduce genes into the skin which would improve its consistency or improve the basic function of the skin.
  • the epidermis which is found on the soles of the feet and the heels and palms of the hands ' is much thicker than the epidermis found in other areas of the body.
  • that epidermis contains a keratin protein of a distinct molecular weight.
  • Another potential use which requires changing the skin's characteristics is in treatment of victims with severe burn. In this case there are several needs for skin to grow very rapidly in order to prevent infection, etc Using this vector system, it is possible to engineer a skin or an epidermis which would be much more suitable for someone who had a severe burn.
  • Another use of the present invention is in the treatment of enzyme defect diseases.
  • the product (polypeptide) encoded by the gene introduced into keratinocytes is not secreted (as are hormones) , but is an enzyme which remains inside the cell.
  • genetic diseases in which the patient lacks a particular enzyme and is not able to metabolize various amino acids or other metabolites.
  • the correct genes for these enzymes could be introduced into a skin transplant; the transplant would then carry out that metabolic function.
  • a genetic disease in which those affected lack the enzyme adenosine deaminase.
  • This enzyme is involved in the degradation of purines to uric acid. It might be possible, using the present invention, to produce a skin graft capable of producing the missing enzyme at sufficiently high levels to detoxify the blood as it passes through the area to which the graft is applied.
  • Epithelial cells having foreign genetic material introduced according to the present inven ⁇ tion can also be used as transdermal drug delivery systems.
  • the drug is applied to the surface of the epidermis and diffuses into the bloodstream through the epidermis.
  • transder ally administered drugs are currently used for prevention of motion sickness.
  • An important limitation to transdermal drug delivery, however, is that the drug must be permeable to the epidermal outer layers; it must be lipid soluble in order to be able to penetrate these layers and be effective. This limitation can be eliminated, however, using epidermis having keratinocytes modified according to the present invention.
  • the drug in its active form, could be made in a lipd soluble - but inactive - form which can pass through the epidermis. It can then be applied to a skin graft having keratinocytes genetically engineered to be able to convert the lipid soluble/inactive drug into a water soluble/ ctive form. The drug could then pass into the bloodstream and produce the desired effect.
  • the present invention also has veterinary applications. It can be used, for example, in delivering substances such as drugs (e.g., anti ⁇ biotics) and hormones to animals, which would otherwise be provided by being incorporated into their fee.d, added to their water or injected period ⁇ ically (e.g., daily or less frequently).
  • drugs e.g., anti ⁇ biotics
  • hormones e.g., hormones
  • Use of the modified epithelial cells of the present invention has the advantage that the tissue formed of the modified epithelial cells can be applied to the animal and will provide quanitites of the encoded protein on an ongoing basis, thus eliminating the need for daily/periodic administration of the substance.
  • the neo gene was introduced into keratinocytes according to the procedure represented in Figure 3.
  • Psi am line producing the neo virus was treated with mitomycin C at 5 micrograms per mil at about 37° for about two hours in Dulbecco's Modified Eagle's Media (DME) without serum.
  • Mitomycin C is an antibiotic which crosslinks DNA and thus renders the cells unable to divide. However, the cells are still capable of producing infectious virus.
  • Psi am cells were treated with mitomycin C on a 10 centimeter dish and washed several times with DME. The suspen ⁇ sion of keratinocytes was plated onto this dish. As a result, the feeder layer used was one producing infectious virus.
  • this feeder layer is G418 resistant (because it has been engineered to contain the neo gene) . Unlike the Psi am line, it does not produce infectious virus. As a result, it can serve as a feeder layer during the selection process. This is referred to as the G418 feeder layer.
  • the disaggregated keratinocytes were plated on this G418 resistant feeder layer and G418 was added at a final concentration in the media of between 0.5 and 1 mgs. per mil. After about two weeks of incubation, those cells which were growing up were resistant to G418; there were no colonies growing up from control cells (uninfected cells) . The G418 resistant cells appeared to be viable normal kera- tinocyte colonies, indicating that the neo gene had been introduced into and expressed in these cells via the retroviral vector.
  • Example 2 Verification of the Introduction of the neo gene into Keratinocytes and Characterization of Keratinocytes Having the neo gene
  • the re ⁇ combinant retrovirus the neo virus
  • a Southern blot analysis was performed on DNA extracted from G418- resistance keratinocytes. Maniatis, T. et al. , In: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (1982) .
  • the DNA was digested by restric ⁇ tion enzymes and a Southern blot hybridization analysis performed.
  • a neo gene was not evident in the uninfected cells.
  • the neo gene in the keratinocytes was different in size from the neo gene present in the feeder layer. It has also been shown that the keratinocytes containing the neo gene are of the normal wild type. This has been demonstrated in several ways.
  • total cell protein was extracted from infected and uninfected cells; fractionated by SDS polyacryla ide elecrophoresis; and visualized by staining by Coomasie ' blue stain.
  • the protein profile of the infected cells was very distinctive of keratinocytes.
  • involucrin is the precursor protein of the cross- linked envelope which is distinctive of terminal differentiation in keratinocytes. It is therefore a distinctive terminal differentiation-marker of keratinocytes.
  • a Western blotting technique demon ⁇ strated the presence of involucrin. Towbin, H. et - al. , Proceedings of the National Academy of Sciences, U.S.A. , 7_6_:4350-4354 (1979) .
  • the G418 resistant keratinocytes were also shown by electron microscopy to have the appearance of normal keratinocytes. The EM pictures show that the G418 resistant keratinocytes colony was five or six cell layers thick. They also reveal the presence of keratin filaments, tonofilaments, and numerous desmosomes—all hallmarks of normal keratinocyte colonies.
  • the G418-resistant keratinocytes have also been grown to confluence, segregated as an intact epi- thelium and transplanted onto athymic or nude mice. This approach demonstrates the ability of the epidermis to continue to differentiate, a process only partially evident in tissue culture. This was carried out in the following manner.
  • the G418 resistant keratinocytes were grown on a Petri dish, using the G418 feeder layer, until adjoining colonies fused and the dish was covered with kera ⁇ tinocytes. Using an enzyme called Dispase, the sheet of epidermal cells was lifted off the dish using the method described by Green and Kehinde. Green, H. and Kehinde, 0., U.S. Patent No. 4,304,866; Green, H.
  • the recombinant retroviral vectors having the neo gene also have a cloning site. This makes it possible, to introduce foreign genetic material into the vector and to have it expressed by keratinocytes cocultivated with the recombinant virus. It is possible to insert the foreign genetic material at the Bam HI cloning site. For example, it is poss ⁇ ible to clone into this site in the retroviral vector a copy of the gene encoding human growth hormone (HGH).
  • HGH is a polypeptide of about 29,000 Daltons which is normally secreted only by the hypothalamus. Keratinocytes capable of making a polypeptide hormone (such as HGH) or another sub ⁇ stance not normally made by such cells could be transplanted onto an individual and serve as a continuous supply system for the hormone or other substance.
  • a Psi am line which produces a recombinant retrovirus having the gene of interest — here, the human growth hormone gene — was constructed in the following way. Plasmid DNA having the recombinant retroviral genome (having the neo gene) was digested with Bam HI. A modified DNA segment encoding a cDNA of human growth hormone was ligated into this site. The plasmid having the proper orientation of the HGH gene was isolated and
  • the Psi am cells producing the HGH-neo recombi ⁇ nant virus construct were used as the feeder layer in cocultivation with disaggregated keratinocytes.
  • the same procedure as described previously for introduction of the neo gene alone was followed to introduce the HGH-neo construct into keratinocytes. That is, after cocultivation with the Psi am cells producing the HGH-neo recombinant virus construct, the keratinocytes were allowed to grow to conflu-. ence;' were disaggregated into keratinocytes; and cocultivated with the NIH 3T3 derived cell line having G418 resistance as previously described.
  • the keratinocytes were assessed for their ability to express the foreign genetic material (in this case, the human growth hormone gene) .
  • An immunoprecipitation method utilizing kera ⁇ tinocytes metabolically labeled with 35 S methionine was used to detect the presence of HGH in the cultured keratinocytes.
  • super- natants were assayed.
  • Antibody specific to HGH was added to the media and allowed to complex with the HGH.
  • the antibody-HGH complex was precipitated using Staphylococus aureus bacteria bugs known to precipitate them. The complex was treated (e.g.
  • HGH-neo recombinant virus uninfected keratinocytes and those infected solely with the neo virus secreted no HGH.
  • Kera ⁇ tinocytes having the HGH-neo recombinant virus are on deposit with the American Type Culture Collection (Rockville, MD) under deposit number CRL8858.
  • radioimrnuno assay which is an excellent method for determining precise amounts of the hormone, it was possible to determine that there was in excess of 30 to 50 nanograms HGH per milliter in the media. This shows that HGH is secreted by the keratinocytes at levels which are similar to physio- logical concentrations. (Human serum normally contains l-5ng HGH per mil.) Thus, this demonstrates that keratinocytes are able to secret a polypeptide hormone which is normally not secreted by keratinocytes and, additionally, that they can secrete this hormone at close to physiological concentrations..
  • Example 4 Introduction of Foreign Genetic Material and Determination of Graft Size Needed for Produc ⁇ tion of Encoded Material at Physiologically Signifi ⁇ cant Levels
  • PTH parathyroid hormone
  • a recombinant retrovirus containing a copy of the gene encoding parathyroid hormone (PTH) was constructed according to the method described in Example 3. The resulting construct was introduced into human keratinocytes by the method, described in Example 3. Keratinocytes resistant to G418 were selected and the population of such cells expanded and allowed to grow to confluence. Confluent cultures of G418 resistant keratinocytes were assayed for synthesis and
  • the rate of secretion is 425ng/ml., as read from the slope of the curve shown in Figure 6.
  • the epithelium in the 75cm 2 flask is equivalent to 20cm2 because, after it is detached from the flask with dispace, the epithelium shrinks to about one-fourth its size. Therefore, the rate per surface area is:
  • a graft 2 9.7cm in size would be needed.
  • a graft of this size would not only provide physiologically signifi ⁇ cant levels of PTH, but also be cosmetically accept ⁇ able to a patient (user) and convenient to graft.
  • genetic modification of keratinocytes necessary to produce a graft of this size would be a reasonably easy task.
  • This invention has industrial applicability in providing hormones, enzymes and drugs to mammals, including humans, in need of such substances. For example, it can be used to provide a continuous supply of a hormone which otherwise would be administered on a periodic basis by injection or oral administration. It is particularly valuable in providing such substances, such as human growth hormone, which are needed for extended periods of time.

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Abstract

Des cellules épithéliales expriment un matériau génétique étranger qui peut être de l'ADN ou de l'ARN absents de cellules épithéliales, de l'ADN ou de l'ARN présents dans des cellules épithéliales mais qui n'y sont pas exprimés à des niveaux biologiquement significatifs, de l'ADN ou de l'ARN présents dans l'épithélium et modifiés pour s'exprimer dans des cellules épithéliales, et tout ADN ou ARN qui peuvent être modifiés pour s'exprimer dans des cellules épithéliales, isolés ou dans n'importe quelle combinaison. En outre, ces cellules épithéliales peuvent exprimer un matériau génétique de codage d'un marqueur sélectionnable par lequel des cellules exprimant le matériau génétique étranger peuvent etre exprimées.
PCT/US1986/001378 1985-07-05 1986-06-30 Cellules epitheliales exprimant un materiau genetique etranger Ceased WO1987000201A1 (fr)

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DE8686904590T DE3681787D1 (de) 1985-07-05 1986-06-30 Expression von fremdem genetischem material in epithelzellen.
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AT86904590T ATE68013T1 (de) 1985-07-05 1986-06-30 Expression von fremdem genetischem material in epithelzellen.

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US4868116A (en) 1989-09-19
CA1339962C (fr) 1998-07-21
JPS63500492A (ja) 1988-02-25
IL79289A (en) 1992-01-15
EP0228458B2 (fr) 1997-10-22
JP2703893B2 (ja) 1998-01-26
ATE68013T1 (de) 1991-10-15
US5698436A (en) 1997-12-16
AU6131086A (en) 1987-01-30
DE3681787D1 (de) 1991-11-07
EP0228458A1 (fr) 1987-07-15
IL79289A0 (en) 1986-09-30
EP0228458B1 (fr) 1991-10-02

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